Fig. 3: GPR174 regulates neovascularization by inhibiting AREG expression in Tregs.

a Heatmap of RNA-seq analysis of the ischemic muscles of WT and Gpr174^−/Y mice 7 days after HLI (n = 3) (q-value < 0.05; |log₂Fc| > 1). b Serum AREG, IL-10, and VEGF protein content in Rag1^−/− mice receiving Tregs 7 days after adoptive transplantation experiments (n = 6 for PBS → Rag1^−/− mice; n = 7 for wild-type Tregs→Rag1^−/− mice; n = 7 for GPR174-deficient Tregs→Rag1^−/− mice). c Relative mRNA levels of Areg, Il-10, and Vegf in Tregs sorted from the gastrocnemius tissues of WT and Gpr174^−/Y mice 7 days after HLI (n = 4). d Representative immunofluorescent images of AREG (red) and DAPI (blue) staining in Tregs isolated from ischemic muscle of WT and Gpr174^−/Y mice 7 days after HLI. Scale bar, 10 μm. e, f Representative images and quantification of vascular sprouting of vessel segments cocultured with AREG neutralizing antibody and Tregs isolated from WT and Gpr174^−/Y mice for 5 days (n = 5). Scale bar, 200 μm. g, h Representative Laser Doppler images and quantification of hindlimb blood perfusion in WT and Gpr174^−/Y mice injected with AAV9-shAreg at indicated times after HLI (n = 7 for WT mice + AAV9-shN and Gpr174^−/Y mice + AAV9-shN; n = 8 for WT mice + AAV9-shAreg and Gpr174^−/Y mice + AAV9-shAreg). i, j Representative immunofluorescent images of CD31 (i) staining and quantification of CD31 (j) in muscle cross sections (n = 6). Scale bar, 50 μm. k, l Representative immunofluorescent images of αSMA (k) staining and quantification of lumen perimeter (l) in muscle cross sections (n = 6). Scale bar, 50 μm. For all statistical plots, the data are presented as mean ± SD. One-way ANOVA with Bonferroni multiple comparisons test in (b). Two-way ANOVA with Bonferroni multiple comparisons test in (c, f, j, l). Two-way repeated measures ANOVA with Sidak’s multiple comparisons test in (h). Source data are provided as a Source Data file.